Vascular Endothelial Growth Factor (VEGF), a pro-angiogenic factor produced by most human solid tumors is important modulator of hematopoiesis. VEGF signaling is crucial for early hematopoietic development. However, recent data including ours, revealed the role of VEGF as inhibitor of immune cell differentiation with profound effect on dendritic (DC) and T cells. We made an important observation that substantial part of VEGF inhibitory effect on DCs is mediated by tyrosine kinase-independent mechanism. Our preliminary data strongly implicate VEGF receptor 1 (VEGFR1) in this process. Identifying specific mechanisms of immunosuppressive effects of VEGF would be essential for the development of efficient therapeutic strategies aimed at immune correction in cancer. The goal of the proposed project is to investigate the mechanism and functional elements of inhibitory signal transduction in this system. The embryonic lethality of most perturbations of VEGF ligand or receptors makes genetic studies of hematopoietic effects more difficult. We have therefore developed in vitro system where genetically modified murine embryonic stem (ES) cells can be differentiated into DCs. We will use murine ES cells knocked out for various receptors and transduced with specific receptor mutants to determine the role of each receptor, their subdomains and signaling partners in this process. Our animal bone marrow transplant studies are designed to elucidate relative contribution of each VEGF receptor and their tyrosine kinases in DC dysfunction in vivo. We hypothesize that VEGFR1 being a major mediator of DC dysregulation transduces substantial part of its inhibitory signaling by mechanism other then tyrosine kinase-dependent, involving juxtamembrane domain, formation of heterodimers, or internalization. We will test this by the following specific aims: 1) Characterize the roles of VEGFR1 non-tyrosine kinase subdomains on DC differentiation and hematopoiesis in vitro. 2) Determine the role of VEGFR1 tyrosine phosphorylation dependent signaling in DC defects in vitro. 3) Assess the role of signaling by each VEGF receptors in mediating VEGF effects on DC development in vivo. Understanding the mechanism and effects of VEGF signaling in hematopoietic cells will lead to greater insight into normal and pathological hematopoiesis, and potential therapeutic interventions aimed at improvement of immune function and highly clinically relevant to cancer and immunotherapy.